KR20010034205A - New pentasaccharides, their methods of production and pharmaceutical compositions containing same - Google Patents

Abstract

The present invention relates to acidic pentasaccharides and pharmaceutically acceptable salts thereof, in which the anionic form has the formula (I).

<Formula 1>

Where

R ₁ represents (C ₁ -C ₃ ) alkyl;

R represents hydrogen or an —SO ₃ —, (C ₁ -C ₃ ) alkyl or (C ₂ -C ₃ ) acyl group;

T represents hydrogen or an ethyl group;

n represents 1 or 2.

Description

New pentasaccharides, preparation method thereof and pharmaceutical composition containing the same {New pentasaccharides, their methods of production and pharmaceutical compositions containing same}

The present invention relates to pentasaccharides, methods for their preparation and pharmaceutical compositions containing them.

Heparin is a polysaccharide of the glycosaminoglycan family known for its anticoagulant properties. As known [I. Bjork and U. Lindahl, Molecular and Cell Biochemistry, 1982, Dr. W. Junk Publishers (Netherlands)], Blood coagulation is a complex physiological phenomenon. Certain stimuli, such as contact activation and tissue factors, stimulate the serial activation of a series of coagulation factors present in plasma. The final stage is the same regardless of the nature of the stimulus. The final step is that activated factor X (Xa) activates factor II (also known as "prothrombin"), and the active form of factor II (factor IIa; also known as "thrombin") causes partial proteolysis of soluble fibrinogen and thus blood Releases insoluble fibrin, one of the main components of coagulation.

In normal physiological conditions, the activity of coagulation factors is regulated by proteins such as antithrombin III (AT III) and heparin cofactor II (HC II), which are also present in plasma. AT III exhibits inhibitory activity against a certain number of coagulation factors and in particular factors Xa and IIa.

Inhibition of factor Xa and factor IIa is the preferred means for obtaining anticoagulant and antithrombotic activity since both factors are involved in the last two stages of coagulation regardless of stimulation.

blood. Pentasaccharides as described in Sinei et al. (P. Sinay et al., Carbohydrate Research 1984, 132 C5) exhibit the minimum heparin sequence required to bind AT III. This compound was obtained by complete chemical synthesis about 15 years ago.

Thereafter, a certain number of synthetic oligosaccharides obtained by complete chemical synthesis and having antithrombotic anticoagulant activity are described in the literature.

EP 0,084,999 discloses derivatives consisting of uronic acid (glucuronic acid or iduronic acid) monosaccharide units with advantageous antithrombotic properties and glucoseamines. In addition to the substituents consisting of hydroxy groups, these compounds contain N-sulfate groups and N-acetyl groups, in which case the anomeric hydroxy groups are substituted with methoxy groups.

European patent application EP 0,165,134 also describes synthetic oligosaccharides having antithrombotic activity. Compounds consisting of uronic acid monosaccharide units and glucoseamines and containing O-sulfate groups at position 3 of the glucoseamine units are also described in European patent application EP 0,301,618. These compounds have strong antithrombotic and anticoagulant properties. European Patent EP 0,454,220 describes uronic acid derivatives and glucose derivatives containing O-alkyl groups or O-sulfate groups as substituents. The latter compounds also have antithrombotic and anticoagulant properties.

Sulfated glycosaminoglycanoid derivatives in which N-sulfate, N-acetate or hydroxy functional groups are substituted with alkoxy, aryloxy, aralkyloxy or O-sulfate groups are described in EP 0,529,175. These compounds also have advantageous antithrombotic properties. The latter compounds are also inhibitors of smooth muscle cell proliferation.

Oligosaccharides, particularly pentasaccharides, similar to the minimum heparin sequence required for binding to AT III are described in Angew. Chem. Int. Ed. Engl. 1993, 32, 3, 434-436. These compounds contain glucuronic acid or glucose units in which hydroxy functional groups are substituted with O-sulfate or O-methyl groups.

Since then, numerous studies on saccharides have been conducted, and it has been pointed out in the literature that the formation of L-iduronic acid unit G plays an important role in the activity of the product. Several structural states of unit G have been described ( ⁴ C ₁ , ¹ C ₄ , ² S ₀ ) and it has been proposed that this structural flexibility is essential for the biological activity of products containing L-duuronic acid [B. Casu, M. Petitou, A. Provasoli and P. Sinay, A New Concept Illustrating the Binding and Biological Properties of Structural Flexibility—Glycosaminoglycans containing Iduronic Acid, Trends Biochem. Sci. 1988 13 221-225.

Surprisingly, it has now been found that by replacing one of the O-alkyl groups with an alkylene bridge, ie by fixing the structure of L-iduronic acid, oligosaccharides with beneficial biological properties are obtained, although they lack structural flexibility. The basis is that, due to its novel structure and potent and unexpected biological properties, the compounds of the present invention differ from other synthetic heparinoids described in the literature. The compound of the present invention is a pentasaccharide wherein L-iduronic acid unit G has a so-called “locked” ² S ₀ structure, and D-iduronic acid unit E optionally has an ethyl group at position 5. These compounds have very high anti-Factor Xa activity and very strong affinity for AT III.

More specifically, the subject of the present invention is a pharmaceutically acceptable salt with pentasaccharide in acidic form and at least one pharmaceutically acceptable cation, whose anionic form is of the formula (I).

Where

R ₁ represents (C ₁ -C ₃ ) alkyl;

R is hydrogen or ^{_{-SO 3 -, (C 1 -C}} 3) alkyl or (C ₂ -C ₃₎ represents an acyl group;

T represents hydrogen or an ethyl group;

n represents 1 or 2.

The present invention includes pentasaccharides in acidic form, in the form of a pharmaceutically acceptable salt. In acidic form the -COO ^- and -SO ₃ ^- functional groups are in the form of -COOH -SO ₃ H, respectively.

The expression "pharmaceutically acceptable salts of pentasaccharides of the invention" refers to pentasaccharides in which one or more -COO ^- and / or -SO ₃ ^- functional groups are ionically bound to a pharmaceutically acceptable metal cation. It is intended to be.

Preferred salts according to the invention are those in which the cation is selected from the cations of the alkali metal, more preferably the cation is Na + or K +.

The subject matter of the present invention is also to prepare a unit G precursor and couple it with a unit H precursor to produce a GH precursor, and finally after coupling the GH precursor with a DEF precursor or after coupling the GH precursor with an EF precursor It is the manufacturing method of the saccharide of this invention characterized by adding D and obtaining a saccharide.

Any precursor of G, H, EF, DEF can be used. This means that the method according to the method can produce all the groups of pentasaccharides with unit G in a commonly submerged structure.

The above method is a preferred method of the present invention. However, the saccharides of the present invention can be prepared by other methods known in sugar chemistry, especially if hydroxyl radicals and optionally carboxyl radicals are present, as described in T. W. Green, Protectors in Organic Synthesis, Wiley, N.Y. 1981 reacting a monosaccharide containing a protecting group with another protected monosaccharide to form a disaccharide, which is then reacted with another protected monosaccharide to form a protected trisaccharide, from the protected sugar It is also possible to form a stream and then obtain a protected saccharide.

The protected pentasaccharide may then be deprotected, optionally sulfated or partially deprotected and then sulfated and then deprotected to prepare the compounds of the present invention.

Such a process is known in carbohydrate chemistry, especially in G. Jaurand et al., Bioorganic and Medicinal Chemistry Letters 1992, 2, 9, 897-900, J. Basten et al., Bioorganic and Medicinal Chemistry Letters 1992, 2, 9, 905-910 and M. Petitou and C.A.A. van Boeckel in "Chemical synthesis of heparin fragment and analogues" 203-210-Process in the chemistry of organic natural products, Ed. Springer Verlag Vienna-N.Y. 1992].

According to the above process, the compounds of the present invention in the form of a salt can be obtained. The compound of the invention in the form of a salt is brought into contact with the cation exchange resin in acidic form to obtain the corresponding acid.

The compound of the invention in acid form can then be neutralized with a base to afford the desired salt.

For this purpose, any inorganic or organic base that produces pharmaceutically acceptable salts can be used.

Sodium hydroxide, potassium hydroxide, calcium hydroxide or magnesium hydroxide are preferably used. The sodium and calcium salts of the pentasaccharide of the present invention are preferred salts.

Compounds that are the subject of the present invention have beneficial pharmacological and biochemical properties. More specifically, they have high anti-Factor Xa activity and high affinity for AT III.

As mentioned above, factor Xa in the coagulation cascade activates prothrombin to thrombin, and proteolytically releases thrombin to insoluble fibrin, a major component of blood coagulation. Therefore, inhibition of Factor Xa is the preferred means for obtaining anticoagulant and antithrombotic activity. When the anti-factor Xa activity of the product of the present invention is evaluated at pH 7 according to the method of Teien AN and Lie M., in Thrombosis Research 1977, 10, 399-410, -Xa activity was shown to be more than that of the synthetic heparinoids already known. D. The affinity for AT III of the pentasaccharide of the invention, whose anion has a structure of formula (I). Atta et al. [D. Atha et al. in Biochemistry 1987, 26, 6454-6461] were measured with a spectrofluorometer under the conditions described. Test results show that the compounds of the present invention have a very high affinity for AT III.

Also, jay. Layer, etc. [J. Reyers et al. in Thrombosis Research 1980, 18, 669-674, the overall antithrombotic activity of these compounds was evaluated using a venous blood flow arrest model in rats and means of induction into thromboplastin. ED ₅₀ of the compound of the present invention is below that of other synthetic heparinoids already known. The compounds of the invention thus have advantageous activity specificities and advantageous anticoagulant and antithrombotic activity.

The compounds of the present invention are useful for preparing pharmaceutical compositions for parenteral administration.

The compounds of the present invention have a very low toxicity which is entirely compatible with their use as a medicament.

The compounds of the present invention are very stable and are therefore particularly suitable as active ingredients in medicine.

The invention also encompasses pharmaceutical compositions which, as active ingredients, contain one of the compounds according to the invention or one of their pharmaceutically acceptable salts, optionally together with one or more inert and suitable excipients.

Within each unit formulation there is an amount of active ingredient suitable for the expected daily dosage. Each unit preparation contains 0.1 to 100 mg, preferably 0.5 to 50 mg of active ingredient.

The compounds according to the invention can be used for anti-platelet aggregation, antithrombotic agents, anticoagulants, such as one or more other active ingredients useful for the desired treatment, such as dipyridamole, aspirin, ticlopidine, clopidogrel or antagonists of IIb / IIIa glycoprotein complexes. It may be used in combination with these.

Pharmaceutical compositions are prepared for administration to mammals, including humans, for the treatment of the diseases described above.

The pharmaceutical compositions thus obtained conveniently come in various forms, for example, injectable or drinking solutions, dragees, plain tablets or gelatin capsules and the like. Injectable solutions are the preferred pharmaceutical form. The pharmaceutical compositions of the present invention may be used for the prophylactic or therapeutic regimen of vascular wall diseases such as atherosclerosis, hypercoagulant conditions observed after tumor surgery or destruction of coagulation induced by bacteria, viruses or enzymatic activators. Particularly useful.

More generally, the saccharides of the present invention can be used to treat pathologies associated with coagulation failure.

The dosage can vary within a wide range depending on the age, weight, health condition, condition and severity of the disease, and route of administration. The dosage is about 0.5 mg to about 1000 mg per day, preferably about 1 to about 100 mg per day, more preferably about 0.5 to about 50 mg per day, for example about 20 mg per day intramuscularly or subcutaneously In one or more batches or at regular intervals, orally orally from about 200 mg to about 1000 mg per day as a daily dose.

Naturally, these dosages are adjustable for individual patients depending on the observed results and previously performed blood assays. Subcutaneous administration is the preferred route.

The subject of the present invention is therefore a pharmaceutical composition comprising one of said compounds as the active ingredient, optionally in combination with another active ingredient. These compositions are prepared so that they can be administered digestively or parenterally.

In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, mucosal, topical or rectal administration, the active ingredient is administered in animal and animal dosage forms in unit dosage form combined with standard pharmaceutical support. It can be administered to humans. Preferred unit dosage forms include oral forms such as oral suspensions, solutions, granules and powders, gelatin capsules, and tablets, sublingual and buccal dosage forms, subcutaneous, intramuscular, intravenous, intranasal or intraocular dosage forms and Rectal dosage forms.

When preparing the solid composition in tablet form, the main active ingredient is mixed with pharmaceutical vehicles such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic and the like. This tablet may be coated with sucrose or other suitable material and may alternatively be treated to have the tablet stopped or delayed activity, thereby continually releasing the desired amount of active material.

Formulations as gelatin capsules are obtained by mixing the active ingredient with a diluent and placing the resulting mixture into soft gelatin or hard gelatin capsules.

The water dispersible powder or granule may contain a dispersing or wetting agent, or an active ingredient in admixture with a suspending agent such as polyvinylpyrrolidone, and a sweetening or flavoring agent.

For rectal administration, suppositories prepared with binders which melt at the temperature of the rectum, for example cocoa butter or polyethylene glycol, are used.

For parenteral, intranasal or intraocular administration, aqueous suspensions, isotonic saline solutions or sterile and injectable solutions containing pharmacologically acceptable dispersing and / or wetting agents such as propylene glycol or butylene glycol This is used.

For mucosal administration, bile salts, hydrophilic polymers such as hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone, pectin, The active ingredient may be formulated in the presence of an accelerator such as starch, gelatin, casein, acrylic acid, acrylic esters and copolymers thereof, vinyl polymers or copolymers, vinyl alcohols, alkoxy polymers, polyethylene oxide polymers, polyethers or mixtures thereof. Can be.

The active ingredient may be formulated in the form of microcapsules, optionally with one or more supports or additives.

The active ingredient may also be in the form of a complex with a cyclodextrin, for example α-, β- or γ-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin or methyl-β-cyclodextrin.

The active ingredient may also be released by a balloon containing it or by an intravascular extender introduced into the blood vessel. The pharmacological efficacy of the active ingredient is therefore not affected.

Subcutaneous administration is the preferred route.

The following method recipes and schemes illustrate the synthesis of various intermediates useful for obtaining the pentasaccharide according to the present invention.

The following abbreviations are used.

TBDMS: tert-butyldimethylsilyl; Lev: leburinyl; Bn: benzyl; Bz: benzoyl; TLC: thin layer chromatography; Olm: trichloroacetimidyl; LSIMS: liquid secondary ion mass spectrometer; ESIMS: electronic spray ionization mass spectrometer; TMS: trimethylsilyl; TSP: sodium trimethylsilyltetraduteropropionate; Tf: triflate; MS: molecular sieves; All: allyl; PMB: p-methoxybenzyl; SE: trimethylsilylethyl.

Dowex , Sephadex , Chelex And Toyopearl Is a registered trademark.

In the following methods, preparations and examples, catalytic coupling of imidate, cleavage of levulinic esters, catalytic coupling of thioglycosides, saponification of p-methoxybenzyl groups, methylation and selective deprotection, The general procedure of deprotection and sulphation of oligosaccharides and polysaccharides by hydrogenolysis of benzyl esters or ethers, saponification or sulphation of esters can be carried out by applying the following general methods to the appropriate intermediates.

The compounds of the present invention are synthesized according to the following various preparation methods.

Scheme 1-Preparation of Precursor GH (17)

1. Preparation of Donor Monosaccharides

2. Preparation and First Conversion of Disaccharides

3. Establishment of binary ring system

Formulation Method 1

6-O-tert-butyldimethylsilyl-1,2-O-isopropylidene-3-O-methyl-α-D-glucofuranose (2)

Diol 1 (10 g, 42.7 mmol) is dissolved in anhydrous dichloromethane (100 ml) and tert-butyldimethylsilyl chloride (7.1 g, 47.3 mmol) and imidazole (5.8 g, 85.3 mmol) are added. The reaction mixture is stirred at room temperature. After 2 hours, the mixture is diluted in dichloromethane and washed with water. The organic layer is dried over magnesium sulfate and concentrated. The residue is purified on a silica gel column (1/9 v / v ethyl acetate / cyclohexane) to afford the desired product 2 (11.9 g, 80%) in syrup form.

[α] _D −34 ° C. (c 1.9 CHCl ₃ ).

Formulation Method 2

6-O-tert-butyldimethylsilyl-1,2-O-isopropylidene-3-O-methyl-5-C-vinyl-α-D-glucofuranose (4)

Oxalyl chloride (3.2 ml, 36.8 mmol) and dimethyl sulfoxide (5.2 ml, 73.4 mmol) are added to anhydrous dichloromethane (40 ml) at −78 ° C. and the mixture is stirred for 30 minutes. Then compound 2 (6.4 g, 18.4 mmol) is added and the mixture is stirred for another 1 hour. Then triethylamine (15.3 ml, 110.0 mmol) is added and after 30 minutes the reaction mixture is diluted with dichloromethane. Penturose compound (3) is obtained by standard reaction finishing and used directly in the subsequent reaction. Crude ketone 3 is dissolved in anhydrous tetrahydrofuran (100 ml) and 1M solution of vinylmagnesium bromide in tetrahydrofuran (28 ml, 27.6 mmol) is added at 0 ° C. After 1 hour, the reaction mixture is diluted (other than ammonium chloride) and washed with water. The organic layer is dried over magnesium sulfate and concentrated, and the residue is purified on a silica gel column (1/9 v / v ethyl acetate / cyclohexane) to afford the desired compound 4 (4.8 g, 70%) in syrup form.

[α] _D −40 ° C. (c 1.3 CHCl ₃ ).

analysis. Calculated: C, 57.72, H, 9.15. Found: C, 57.77, H, 9.23.

Preparation Method 3

1,2,4,6-tetra-O-acetyl-3-O-methyl-5-C-vinyl-β-D-glucopyranose (6)

Compound 4 (3.5 g, 9.4 mmol) was dissolved in water (50 ml); IR-120 resin (1 g) is added thereto and the mixture is heated at 80 ° C. for 6 hours. The resin is filtered off and the filtrate is concentrated. Crude product 5 is acetylated with acetic anhydride (12 ml) and pyridine (13 ml). Excess acetic anhydride is destroyed with methanol and the solvent is concentrated. The residue is extracted with water and dichloromethane. The organic layer is dried over magnesium sulfate, concentrated and purified on a silica gel column (3/2 v / v ethyl acetate / cyclohexane) to give tetraacetate 6 in solid form (75%, 2.7 g). m.p. = 50 ° C.

[α] _D −84 ° C. (c 1.6 CHCl ₃ ).

analysis. Calculated: C, 52.47, H, 6.19. Found: C, 52.51, H, 6.19.

CI-MS: 406 (M + NH ₄ ), 389 (M + 1).

Formulation Method 4

Methyl 2,3,6-tri-O-benzyl-4-O- (2,4,6-tri-O-acetyl-3-O-methyl-5-C-vinyl-β-D-glucopyranosyl) -α-D-glucanoside (8)

Compound 6 (1.6 g, 4.1 mmol) and Compound 7 (2.1 g, 4.5 mmol) (PJ Garegg and H. Hultberg, Carbohydr.Res. 1981, 93, C10) were dissolved in anhydrous dichloromethane (50 ml) and separated Molecular sieve (4.0 g) is added. The reaction mixture is stirred at rt for 1 h, then TMSOTf (0.95 ml, 5.2 mmol) is added at -78 ° C. The reaction mixture is left to soften until it is warmed to room temperature. After 2 h the reaction mixture was neutralized with triethylamine and filtered through Celite; Wash the filtrate with water. The organic layer is dried over magnesium sulfate and concentrated, and the residue is purified on a silica gel column (4/1 v / v ethyl acetate / cyclohexane) to afford the desired compound 8 (2.77 g, 85%) in solid form. m.p. = 47 ° C.

[α] _D −36 ° C. (c 0.6 CHCl ₃ ).

analysis. Calculated: C, 65.14, H, 6.61. Found: C, 65.09, H, 6.70.

Recipe 5

Methyl 2,3,6-O-tri-O-benzyl-4-O- (4,6-O-isopropylidene-3-O-methyl-5-C-vinyl-β-D-glucopyranosyl) -α-D-glucopyranoside (10)

Compound 8 (2.7 g, 3.4 mmol) is dissolved in methanol (40 ml). Sodium (catalyst) is added at 0 ° C. and the mixture is stirred at room temperature for 3 hours. The solvent is concentrated and the residue 9 is dissolved in anhydrous acetone (40 ml) and 2,2-dimethoxypropane (2 ml) and p-toluenesulphonic acid (catalytic) are added. The reaction mixture is stirred overnight at room temperature. The solvent is removed by evaporation and the residue is taken up in chloroform and washed with water. The organic layer was dried over magnesium sulfate and concentrated and the residue was purified on a silica gel column (1/1 v / v ethyl acetate / cyclohexane) 4 ', 6'-isopropylidene-O-derivative 10 (1.7 g, 70%) Is obtained in solid form. m.p. = 55 ° C.

[α] _D −13 ° C. (c 0.8 CHCl ₃ ).

analysis. Calculated Value: C, 67.97, H, 7.13. Found: C, 67.87, H, 7.16.

CI-MS: 707 (M + 1), 724 (M + NH ₄ ).

Preparation Method 6

Methyl 2,3,6-tri-O-benzyl-4-O- (4,6-O-isopropylidene-3-O-methyl-5-C-vinyl-β-D-mannopyranosyl) -α -D-glucopyranoside (12)

Oxalyl chloride (0.35 ml, 4.0 mmol) and anhydrous DMSO (0.57 ml, 8.0 mmol) are stirred in anhydrous dichloromethane (10 ml) at −78 ° C. for 30 minutes. Compound 10 (1.4 g, 2.0 mmol) in anhydrous dichloromethane (10 ml) was added to this solution and stirring continued for an additional 45 minutes. The reaction mixture is neutralized by addition of anhydrous triethylamine (1.7 ml, 12.0 mmol) and diluted with dichloromethane. After washing with water, the organic layer is dried over magnesium sulfate and concentrated. The residue 11 is used directly in the subsequent reaction without purification. Ketone 11 is dissolved in anhydrous tetrahydrofuran (15 ml) and a 1N solution of super hydride in tetrahydrofuran (4 ml, 4.0 mmol) is added at -78 ° C. The reaction mixture is stirred for 1 hour at room temperature and 5% sodium hydroxide (2 ml) and hydrogen peroxide (1 ml) are added. The solvent is removed by evaporation and the residue is dissolved in ethyl acetate and washed with water. The organic layer is dried over magnesium sulfate and concentrated. The residue is purified by chromatography (2/1 v / v ethyl acetate / cyclohexane) to give compound 12 (1.0 g, 70%).

[α] _D −11 ° C. (c 0.5 CHCl ₃ ).

CI-MS: 724 (M + 18), 707 (M + 1).

Recipe 7

Methyl 2,3,6-tri-O-benzyl-4-O- (2-O-acetyl-3-O-methyl-5-C-vinyl-β-D-mannopyranosyl) -α-D-glu Copyranoside (14)

Compound 12 (940 mg, 1.3 mmol) is dissolved in pyridine (3 ml) and acetic anhydride (0.3 ml). It is stirred for 3 hours at room temperature. Excess pyridine and acetic anhydride are concentrated and the residue 13 is used directly for deprotection of isopropylidene with 80% acetic acid (5 ml) at 60 ° C. for 2 hours. Excess acetic acid is evaporated off and the residue is purified by silica gel column chromatography (4/1 v / v ethyl acetate / cyclohexane) to give diol 14 (660 mg, 70%) in solid form. m.p. = 53 ℃

[α] _D −10 ° C. (c 0.8 CHCl ₃ ).

CI-MS: 709 (M + 1), 726 (M + 18).

Preparation Method 8

Methyl 2,3,6-tri-O-benzyl-4-O- (2-O-acetyl-3-O-methyl-6-O-tosyl-5-C-vinyl-β-D-mannopyranosyl) -α-D-glucopyranose (15)

Compound 14 (600 mg, 0.9 mmol) is dissolved in pyridine (3 ml) and tosyl chloride (240 mg, 1.3 mmol) is added. It is stirred for 3 hours at room temperature. The solvent is evaporated off and the residue is diluted with chloroform and washed with water. The organic layer is dried over magnesium sulfate and concentrated. The residue was purified by silica gel column chromatography (1/1 v / v ethyl acetate / cyclohexane) to obtain tosyl compound 15 (279 mg, 80%) in the form of syrup.

[α] _D −26 ° C. (c 0.8 CHCl ₃ ).

Recipe 9

Methyl 2,3,6-tri-O-benzyl-4-O- (2,6-anhydro-3-O-methyl-5-C-vinyl-β-D-mannopyranosyl) -α-D- Glucopyranoside (16)

Compound 15 (550 mg, 0.6 mmol) is dissolved in ethanol (3 ml) and 0.1N sodium hydroxide ethanol solution (5 ml) is added. It is stirred at 70 ° C. for 3 hours, then neutralized with IR-120 resin (H + form) and filtered through Celite. After concentration, the residue was purified by silica gel column chromatography (1/1 v / v ethyl acetate / cyclohexane) to give compound 16 (292 mg, 70%) in syrup form.

[α] _D + 13 ° C. (c 0.5 CHCl ₃ ).

CI-MS: 666 (M + 18).

Preparation Method 10

Methyl 2,3,6-tri-O-benzyl-4-O- (benzyl-3-O-methyl-2-O-5-C-methylidene-α-L-idopyranuronate) -α-D -Glucopyranoside (17)

Compound 16 (260 mg, 0.4 mmol) was dissolved in dichloromethane (20 ml), the solution was stirred at −78 ° C. and ozone was passed through bubbling for 30 seconds. The solution turns light yellow. Dimethyl sulfide is added and the reaction mixture is washed with water. The organic layer was dried over magnesium sulphate and concentrated and used directly in the subsequent reaction without further purification. The crude aldehyde is dissolved in tert-butanol (16 ml) and 2-methyl-2-butene (5 ml) and water (16 ml) are added. Then NaH ₂ PO ₄ (700 mg) and NaClO ₂ (700 mg) are added sequentially. The suspension is stirred vigorously overnight at room temperature, diluted with water and extracted with ethyl acetate. The organic layer is dried over magnesium sulphate and concentrated and the subsequent reaction is carried out directly. The crude acid is dissolved in dimethylformamide (25 ml) and tetrabutylammonium iodide (0.7 g, 2.0 mmol), potassium bicarbonate (0.25 g, 2.5 mmol) and benzyl bromide (0.250 ml, 2.1 mmol) are added. . The reaction mixture is stirred at room temperature for 5 hours. The reaction mixture is extracted with water and diethyl ether. The ether layer was concentrated to dryness with magnesium sulfate, and the residue was purified by silica gel column chromatography (2/1 v / v ethyl acetate / cyclohexane) to obtain a derivative 17 (236 mg, 80%) in the form of a syrup.

CI-MS: 774 (M + 18).

Scheme 2: Condensation, deprotection and sulfated of imidate DEF (18) with GH (17).

Preparation Method 11

Methyl O- (6-O-acetyl-2,3,4-tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (benzyl 2,3-di-O-methyl -β-D-glucopyranosyluronate- (1 → 4) -O- (3,6-di-O-acetyl-2-O-benzyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,6-Anhydro-5-C-benzyloxy-carbonyl-3-O-methyl-β-D-mannopyranosyl)-(1 → 4) -2,3,6-tri- O-benzyl-α-D-glucopyranoside (19)

Imidate 18 (Van der Heijden et al., Abstr. 9th, Eur.Carbohydr.Symp.Utrecht, July 6-11, 1997; A74, p154) (81 mg, 78.2 μmol), receptor 17 (65 mg, 86.0 μmol ) And fine molecular sieves (70 mg, 4 cc) are stirred in 1/2 (v / v) dichloromethane / diethylether mixture (2.4 ml) under an inert atmosphere.

The reaction mixture is stirred for 30 minutes and NaHCO ₃ is added at the neutralization point. After filtration and concentration, the residue was purified on a Sephadex LH 20 gel (1/1 v / v dichloromethane / ethanol) column and then on a silica gel (1/1 v / v ethylacetate / cyclohexane) column to give Compound 19 ( 86 mg, 67%).

[α] _D + 66 ° C. (c 1.0 CH ₂ CH ₂ ).

¹ H NMR is shown in Table 1 below.

Preparation 12

Methyl O- (2,3,4-Tri-O-methyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl-β-D-glucopyra Nosyluronic acid- (1 → 4) -O- (α-D-glucopyranosyl)-(1 → 4) -O- (2,6-anhydro-5-C-carboxy-3-O-methyl -β-D-mannopyranosyl)-(1 → 4) -α-D-glucopyranoside (20)

M. Petittu and Mr. a. a. Van Beckell [M. Petitou and C.A.A. Boechel, Progress in the Chemistry of Organic Natural Products, published by W. Herz et al. Vienna, Springer-Verlag, New York, 1992, 143-210.

A solution of compound 19 (49 mg, 30.0 μmol) in acetic acid (3 ml) is stirred for 12 h under hydrogen (3.5 MPa) at 40 ° C. in the presence of 5% palladium (73 mg, 35 b) on activated carbon. The mixture is filtered through Celite, concentrated and co-distilled with water (4 x 5 ml). The residue is dissolved in 1M aqueous sodium hydroxide solution (3 ml) and heated at 55 ° C. for 3 hours. The solution is cooled, passed through a Sephadex 625F column (170 ml) and eluted with water. Fractions containing compound 20 are passed through a Dowex H ⁺ resin column. Concentrate the eluate to afford compound 20 (25 mg, 86%).

[α] _D + 105 ° C. (c 1.0 H ₂ O).

¹ H NMR is shown in Table 1 below.

Scheme 3: Preparation of donor EF, disaccharide 29.

Preparation Method 13

Methyl O- (4,6-O-isopropylidene-2,3-di-O-methyl-5-C-vinyl-β-D-glucopyranosyl)-(1 → 4) -2,3,6 -Tri-O-benzyl-α-D-glucopyranoside (22)

Methyl-O- (4,6-O-isopropylidene-3-O-methyl-5-C-vinyl-β-D-glucopyranosyl)-(1 → 4) -2,3,6-tri- Sodium hydroxide in a solution of O-benzyl-α-D-glucopyranoside 10 (6.11 g, 8.65 mmol) and methyl iodide (0.80 ml, 13.0 mmol) in N, N-dimethylformamide (9.00 ml) (0.31 g, 13.0 mmol) is added at 0 ° C. The mixture is stirred for 2 hours (TLC), methanol is added and the reaction mixture is poured into water. The mixture is extracted with ethyl acetate and the extract is washed with water, dried and concentrated. The residue is purified on a silica column (3/2 v / v cyclohexane / diethyl ether) to give compound 22 (5.92 g, 88%).

TLC: Rf = 0.28, (3.2 v / v cyclohexane / diethyl ether)

Recipe 14

Methyl O- (4,6-O-isopropylidene-2,3-di-O-methyl-5-C-ethyl-β-D-glucopyranosyl)-(1 → 4) -2,3,6 -Tri-O-benzyl-α-D-glucopyranoside (23)

To a solution of compound 22 (5.80 g, 8.04 mmol) in ethyl acetate (400 ml) is added platinum oxide (160 mg). Introduce hydrogen. The mixture is left under stirring for 40 minutes (TLC). Filtration and evaporation yields compound 23.

TLC: Rf = 0.60, (4.1 v / v toluene / ethylacetate).

Formula 15

Methyl O- (2,3-di-O-methyl-5-C-ethyl-β-D-glucopyranosyl)-(1 → 4) -2,3,6-tri-O-benzyl-α-D -Glucopyranoside (24)

Crude Compound 23 is dissolved in 70% acetic acid (60 ml) and stirred at 80 ° C. for 2 hours. The mixture is concentrated in vacuo and co-evaporated with toluene to give compound 24.

TLC: Rf = 0.52, (4/1 v / v dichloromethane / methanol)

Preparation 16

Methyl O- (benzyl 2,3-di-O-methyl-5-C-ethyl-β-D-glucopyranosyluronate)-(1 → 4) -2,3,6-tri-O-benzyl- α-D-glucopyranoside (25)

2,2,6,6-tetramethyl-1-piperidyloxy (17.5 mg), sodium bicarbonate solution (17.5 ml), potassium bromide (87 mg) and tetrabutylammonium chloride (115.5 mg) To a solution of compound 24 (5.75 g) in hydrofuran (28 ml). The mixture is cooled to 0 ° C. and a mixture of saturated sodium chloride solution (17 ml), saturated sodium bicarbonate solution (8.70 ml) and sodium hypochlorite (1.3M, 20 ml) is added over 15 minutes. After stirring for 1 hour, the mixture is diluted with water and extracted three times with dichloromethane. The organic layer is washed with aqueous sodium chloride solution, dried over magnesium sulfate, filtered and evaporated to dryness to afford a crude acid derivative.

This acid derivative is dissolved in N, N-dimethylformamide (107 ml) under a nitrogen atmosphere. Potassium hydrogen carbonate (4.10 g) and benzyl bromide (9.70 ml) are added and the mixture is stirred for 16 hours. Ethyl acetate and water are added and the organic layer is concentrated after extraction. Purification by chromatography on silica gel affords 3.81 g of compound 25 (60% yield from compound 23).

[α] _D +24 (c 0.15, dichloromethane).

Preparation 17

Methyl O- (benzyl-5-C-ethyl-4-O-levulinyl-2,3-di-O-methyl-β-D-glucopyranosyluronate)-(1 → 4) -2,3 , 6-tri-O-benzyl-α-D-glucopyranoside (26)

Compound 25 (3.51 g, 4.46 mmol) is dissolved in anhydrous dioxane (45 ml). Revulinic Acid (1.00 g, 8.93 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1.70 g, 8.93 mmol) and 4-dimethylaminopyridine (0.11 g, 8.93 mmol Add). The mixture is left under stirring for 16 hours and extracted with ethyl acetate. The extract is washed successively with 5% aqueous potassium hydrogen sulphate solution, water, saturated aqueous sodium hydrogen carbonate solution and water, dried and concentrated. The residue is purified on a silica column (2/1 and then 3/2 v / v cyclohexane / ethyl acetate) to give pure compound 26 (3.64 g, 85%).

[a] _D +26 (c = 0.9, dichloromethane).

Preparation 18

O- (benzyl-5-C-ethyl-4-O-levulinyl-2,3-di-O-methyl-β-D-glucopyranosyluronate)-(1 → 4) -1,3, 6-Tri-O-acetyl-2-O-benzyl-D-glucopyranose (27)

Compound 26 (3.35 g, 3.78 mmol) is dissolved in acetic anhydride (22 ml). The solution is cooled to −20 ° C. and 22 ml of sulfuric acid ice solution (1 ml of sulfuric acid in 10 ml of acetic anhydride) are added. The mixture is diluted with ethyl acetate and dried and concentrated by washing with saturated aqueous sodium hydrogen carbonate solution and with water. The residue is purified on a silica column (1/1 v / v cyclohexane / ethyl acetate) to give compound 27 (2.20 g, 65.5%).

TLC: Rf = 0.24 (1/1 v / v cyclohexane / ethyl acetate)

Preparation 19

O- (benzyl-5-C-ethyl-4-O-levulinyl-2,3-di-O-methyl-β-D-glucopyranosyluronate)-(1 → 4) -3,6- Di-O-acetyl-2-O-benzyl-D-glucopyranose (28)

Benzylamine (11 ml, 101.4 mmol) is added to a solution of compound 27 (2.18 g, 2.67 mmol) in tetrahydrofuran (50 ml). It is left under stirring for 4 hours. Extract with ethyl acetate, the extract is washed with 1 M aqueous hydrochloric acid solution (102 ml) and water, dried and concentrated. The residue was purified on a silica column (1/1 v / v toluene / ethyl acetate) to give a mixture of compound 28 (α / β = 50/50) (1.33 g, 65%).

TLC: Rf = 0.22 (1/1 v / v toluene / ethyl acetate)

Preparation 20

O- (benzyl-5-C-ethyl-4-O-levulinyl-2,3-di-O-methyl-β-D-glucopyranosyluronate)-(1 → 4) -3,6- Di-O-acetyl-2-O-benzyl-D-glucopyranosyl trichloroatethymidate (29)

Compound 28 (1.32 g, 1.71 mmol) is dissolved in dichloromethane (34 ml) and trichloroacetonitrile (0.87 ml, 8.50 mmol) and cesium carbonate (0.90 g, 2.73 mmol) are added under argon. The mixture is left stirring (TLC) for 2 hours and filtered. The residue is purified on a silica column (3/2 v / v cyclohexane / ethyl acetate) to give a mixture of imidate 29 (α / β = 85/15) (1.20 g, 77%).

TLC: Rf = 0.36 (1/2 v / v cyclohexane / ethyl acetate)

Scheme 4: Preparation of receptor tetrasaccharide 31

Preparation 21

Methyl O- (benzyl 5-C-ethyl-4-O-levulinyl-2,3-di-O-methyl-β-D-glucopyranosyluronate- (1 → 4) -O- (3, 6-di-O-acetyl-2-O-benzyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,6-anhydro-5-C-benzyloxycarbonyl-3- O-Methyl-β-D-mannopyranosyl)-(1 → 4) -2,3,6-tri-O-benzyl-α-D-glucopyranoside (30)

In the presence of 4 ′ molecular sieve (1.93 g), imidate 29 (1.19 g, 1.29 mmol) and methyl 2,3,6-tri-O-benzyl-4- (benzyl 3-O-methyl-2-O-5 -C-methylidene-α-L-idopyranuronate) -α-D-glucopyranoside 17 (1.02 g, 1.35 mmol) in a solution in toluene (40 ml) in dichloromethane (1M, 0.19 ml) A solution of tert-butyldimethylsilyl triflate is added under -20 ° C argon. After 30 minutes (TLC), a solution of tert-butyldimethylsilyl triflate (1M, 0.19 ml) in dichloromethane is added again. After 30 minutes (TLC), sodium bicarbonate solid is added. The solution is filtered, washed with water, dried and evaporated to dryness. The residue is purified by Sephadex LH20 column chromatography and silica column chromatography (2/1 v / v toluene / ethyl acetate) to give pure tetrasaccharide 30-α (1.14 g, 58%).

[α] _D +47 (c = 0.21, dichloromethane).

Recipe 22

Methyl O- (benzyl 5-C-ethyl-2,3-di-O-methyl-β-D-glucopyranosyluronate- (1 → 4) -O- (3.6-di-O-acetyl-2- O-benzyl-α-D-glucopyranosyl)-(1 → 4) -O- (2,6-anhydro-5-C-benzyloxycarbonyl-3-O-methyl-β-D-mannopira Nosyl)-(1 → 4) -2,3,6-tri-O-benzyl-α-D-glucopyranoside (31)

Compound 30 (1.13 g, 0.75 mmol) is dissolved in 2/1 (v / v) ethanol / toluene mixture (150 ml) and hydrazine acetate (0.35 g, 3.73 mmol) is added. The mixture was left stirring (TLC) for 1 hour and concentrated. The residue is purified by silica column chromatography (3/2 (v / v) toluene / ethyl acetate) to give compound 31 (0.816 g, 83%).

[a] _D +35 (c = 1.01, dichloromethane).

Scheme 5: Coupling, Deprotection and Sulfation of Glycoside EFGH (31) with Glycosyl Donor D (32)

Preparation 23

Methyl O- (6-O-acetyl-2,3,4-tri-O-methyl-α-D-glucopyranosyl- (1 → 4) -O- (benzyl 5-C-ethyl-2,3- Di-O-methyl-β-D-glucopyranosyluronate- (1 → 4) -O- (3,6-di-O-acetyl-2-O-benzyl-α-D-glucopyranosyl)- (1 → 4) -O- (2,6-Anhydro-5-C-benzyloxycarbonyl-3-O-methyl-β-D-mannopyranosyl)-(1 → 4) -2,3, 6-Tri-O-benzyl-α-D-glucopyranoside (33)

6-O-acetyl-2,3,4-tri-O-methyl-D-glucopyranose trichloroacetimidate 32 (34.7 mg, 0.0245 mmol) (P. Westerduin, et al. BioOrg. Med. Chem. , 1994, 2, 1267) and glycosyl receptor 31 (80 mg, 0.056 mmol) are treated according to Preparation 21. The compound was purified by Sephadex LH-20 column chromatography (1/1 (v / v) dichloromethane / ethanol) and silica column chromatography (3/2 (v / v) diisopropyl ether / ethyl acetate) to obtain derivative 33. (54.6 mg, 58%) is obtained.

[α] _D +55 (c = 1, dichloromethane).

Recipe 24

Methyl O- (6-O-acetyl-2,3,4-tri-O-methyl-α-D-glucopyranosyl- (1 → 4) -O- (5-C-ethyl-2,3-di -O-methyl-β-D-glucopyranosiluronic acid- (1 → 4) -O- (3,6-di-O-acetyl-α-D-glucopyranosyl)-(1 → 4)- O- (2,6-Anhydro-5-C-carboxy-3-O-methyl-β-D-mannopyranosyl)-(1 → 4) -α-D-glucopyranoside (34)

A solution of compound 33 (40 mg, 0.024 mmol) in acetic acid (2 ml) is stirred under hydrogen atmosphere for 16 hours in the presence of 10% palladium (80 mg) on activated carbon and filtered. The filtrate is concentrated to give compound 34.

Recipe 25

Methyl O- (2,3,4-tri-O-methyl-α-D-glucopyranosyl- (1 → 4) -O- (5-C-ethyl-2,3-di-O-methyl-β -D-glucopyranosiluronic acid- (1 → 4) -O- (α-D-glucopyranosyl)-(1 → 4) -O- (2,6-anhydro-5-C-carboxy- 3-O-Methyl-β-D-mannopyranosyl)-(1 → 4) -α-D-glucopyranoside (35)

5 M aqueous sodium hydroxide solution (216 μl) is added to a solution of crude compound 34 (866 μl) in methanol. After 25 hours, water is introduced and the reaction mixture is passed through a column of Sephadex G-25 gels (2 x 38 cm) and eluted with water. The eluate is concentrated, then passed through a Dowex 50 H + column (2 ml) and lyophilized. In this step, ¹ H NMR is used to check that all protecting groups have been removed.

<Example 1>

Methyl O- (2,3,4-Tri-O-methyl-6-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl- β-D-glucopyranosiluronic acid)-(1 → 4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,6-Anhydro-3--3-methyl-α-L-idopyranosiluronic acid)-(1 → 4) -2,3,6-tri-O-sulfo-α-D-glucopy Ranozid (21)

Seed. a. a. Van Beckell and M. Petittu [C.A.A. van Boeckel and M. Petitou, Angew. Chem. Int. Ed. Engl., 1993, 32, 1671-1690].

A solution of triethylamine / sulfur trioxide complex (164 mg, 0.90 μmol) in compound 20 (20 mg, 20.7 μmol) and dimethylformamide (2 ml) was shielded from light and kept at 55 ° C. for 18 hours 30 minutes. Heat. The reaction mixture is cooled to room temperature and then diluted with 0.2 M aqueous NaCl solution. The solution is then placed on top of a Sephadex G25F (170 ml) column and eluted with 0.2M aqueous NaCl solution.

Fractions containing saccharides are concentrated and the same column is eluted with water to remove brine. After freeze drying, compound 21 is obtained (30.5 mg, 85%).

[α] _D + 49 ° C. (c = 0.63, H ₂ O).

¹ H NMR is shown in Table 2 below.

<Example 2>

Methyl O- (2,3,4-Tri-O-methyl-6-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (5-C-ethyl-2,3- Di-O-methyl-β-D-glucopyranosiluronic acid)-(1 → 4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,6-anhydro-5-C-carboxy-3-O-methyl-β-D-mannopyranosyl)-(1 → 4) -2,3,6-tri-O Sulfo-α-D-glucopyranoside, sodium salt (36)

Triethylamine / sulfur trioxide complex (91 mg) is added to a solution of crude compound 35 in dimethylformamide (1.3 ml). After 20 hours at 55 ° C., the solution is placed on top of a Sephadex G-25 (2 × 38 cm) column and eluted with a 0.2 M NaCl aqueous solution.

Fractions containing saccharides are concentrated and the same column is eluted with water to remove brine. After lyophilization, compound 36 is obtained (21.9 mg, 52% from compound 33).

¹ H NMR is shown in Table 3 below.

In the same manner as in Examples 1 and 2, the compounds 37 to 40 of the following Examples 3 to 6 were prepared. The ¹ H NMR spectrum obtained for these compounds is consistent with the structure shown below.

<Example 3>

Methyl O- (2,3,4-Tri-O-methyl-6-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl- β-D-glucopyranosiluronic acid)-(1 → 4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,6-Anhydro-5-C-carboxy-3-O-methyl-β-D-mannopyranosyl)-(1 → 4) -2-O-sulfo-3,6-di-O-methyl -α-D-glucopyranoside, sodium salt (37)

[α] _D +51 ° C (c = 0.48, H ₂ O).

<Example 4>

Methyl O- (2,3,4-Tri-O-methyl-6-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl- β-D-glucopyranosiluronic acid)-(1 → 4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,6-Anhydro-5-C-carboxy-3-O-methyl-β-D-mannopyranosyl)-(1 → 4) -2,3-di-O-sulfo-6-O-methyl -α-D-glucopyranoside, sodium salt (38)

[a] _D + 57 ° C (c = 0.28, H ₂ O).

<Example 5>

Methyl O- (2,3,4-Tri-O-methyl-6-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl- β-D-glucopyranosiluronic acid)-(1 → 4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,6-Anhydro-5-C-carboxy-3-O-methyl-β-D-mannopyranosyl)-(1 → 4) -2,6-di-O-sulfo-3-O-methyl -α-D-glucopyranoside, sodium salt (39)

[α] _D + 53 ° C. (c = 0.3, H ₂ O).

<Example 6>

Methyl O- (2,3,4-Tri-O-methyl-6-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O-methyl- β-D-glucopyranosiluronic acid)-(1 → 4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,7-Anhydro-5-C-carboxy-6-deoxy-3-O-methyl-β-D-mannoheptopyranosyl)-(1 → 4) -2,3,6-tri-O Sulfo-α-D-glucopyranoside, sodium salt (40)

[α] _D 49 ° C. (c = 0.25, H ₂ O).

Claims (3)

Pentasaccharides in acidic form and pharmaceutically acceptable salts thereof, wherein the anionic form has the formula (I): <Formula 1> Where R ₁ represents (C ₁ -C ₃ ) alkyl; R is hydrogen or ^{_{-SO 3 -, (C 1 -C}} 3) alkyl or (C ₂ -C ₃₎ represents an acyl group; T represents hydrogen or an ethyl group; n represents 1 or 2. The pentasaccharide according to claim 1, which is in the form of sodium salt and potassium salt. The method according to claim 1 or 2, (i) Methyl O- (2,3,4-tri-O-methyl-6-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,6-anhydro- 5-C-carboxy-3-O-methyl-β-D-mannopyranosyl)-(1 → 4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl) -(1 → 4) -O- (2,6'-anhydro-3-O-methyl-α-L-idopyranosyluronic acid)-(1 → 4) -2,3,6-tri- O-sulfo-α-D-glucopyranoside, sodium salt, (ii) Methyl O- (2,3,4-Tri-O-methyl-6-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (5-C-ethyl-2 , 3-di-O-methyl-β-D-glucopyranosiluronic acid)-(1 → 4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl) -(1 → 4) -O- (2,6-anhydro-5-C-carboxy-3-O-methyl-β-D-mannopyranosyl)-(1 → 4) -2,3,6- Tri-O-sulfo-α-D-glucopyranoside, sodium salt, (iii) methyl O- (2,3,4-tri-O-methyl-6-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,3-di-O -Methyl-β-D-glucopyranosiluronic acid)-(1 → 4) -O- (2,3,6-tri-O-sulfo-α-D-glucopyranosyl)-(1 → 4) -O- (2,6-Anhydro-5-C-carboxy-3-O-methyl-β-D-mannopyranosyl)-(1 → 4) -2-O-sulfo-3,6-di- O-methyl-α-D-glucopyranoside, sodium salt Saccharides selected from.